Cell culture has generated considerable interest in recent years due to the revolution in genetic engineering and biotechnology. Cells are cultured to make proteins, receptors, vaccines, and antibodies for therapy, research, and for diagnostics.
One limitation to the use of this technology is the high cost of operation. Traditionally, cell culture has been operated in a batch mode. In batch operation, the bioreactor is seeded with a small amount of cells and the cells are grown to high density. The cells secrete the product of interest and eventually die due to lack of nutrients at which point the culture is harvested. This method has several drawbacks: firstly, a large fraction of nutrients are wasted in simply growing up cells and are not used directly for making the product; secondly, product formation is often inhibited due to the buildup of toxic metabolic byproducts; and lastly critical nutrients are often depleted leading to low cell densities and consequently lower product yields.
It has long been recognized that perfusion culture offers better economics. In this operation, cells are retained in the bioreactor, and the product is continuously removed along with toxic metabolic byproducts. Feed, containing nutrients is continually added. This operation is capable of achieving high cell densities and more importantly, the cells can be maintained in a highly productive state for weeks and even months. This achieves much higher yields and reduces the size of the bioreactor necessary. It is also a useful technique for cultivating primary or other slow growing cells.
The idea of perfusion has been known since the beginning of the century, and has been applied to keep small pieces of tissue viable for extended microscopic observation. The technique was initiated to mimic the cells milieu in vivo where cells are continuously supplied with blood, lymph, or other body fluids. Without perfusion, cells in culture go through alternating phases of being fed and starved, thus limiting full expression of their growth and metabolic potential.
The current use of perfused culture is in response to the challenge of growing cells at high densities (i.e., 0.1-5×108 cells/ml). In order to increase densities beyond 2-4×106 cells/ml, the medium has to be constantly replaced with a fresh supply in order to make up for nutritional deficiencies and to remove toxic products. Perfusion allows for a far better control of the culture environment (pH, pO2, nutrient levels, etc.) and is a means of significantly increasing the utilization of the surface area within a culture for cell attachment.
Perfusion cell culture has long been used as a method of achieving higher cell densities and increased culture length when compared to batch culture methods. The greater cell density and longer culture life can result in better yields of secreted products, more efficient use of media and the ability to generate large numbers of cells in small volumes. The greatest challenge of perfusion cell culture has always been the need to contain the cells within the culture without reducing cell viability. Filters have traditionally been used to contain the cells, and elaborate methods have been devised to prevent the filters from fouling and shortening the length of the perfusion culture. All of these steps add substantial cost to the manufacturing process.
The U.S. Pat. No. 5,443,985 suggests that to prevent the clogging of the perfusion filter, it should be placed in the upper part of an inclined bioreactor where it is less likely to encounter the cells that might block the filter.
The U.S. Pat. No. 6,544,788 to Singh discloses a perfusion filter with neutral buoyancy that allows this ‘lily pad’ filter to float just under the surface of the media where the wave action that mixes and oxygenates the culture helps wash the filter. The gentle washing prevents the filter from clogging and extends the length of the perfusion culture while maintaining a low shear environment for the cells. This invention is of little use in bioreactors where the suspension culture is maintained rather homogenous throughout the bioreactor obviating any advantage of keep the filter floating at the surface where the cell count is expected to be the lowest; this may be of some value in rocking platform bioreactors like the GE Wave Bioreactor but for large scale suspension culture where the cells are kept in uniform suspension, both of the above prior art disclosures are of little practical value.
The development of a perfused packed-bed reactor using a bed matrix of a non-woven fabric has provided a means for maintaining a perfusion culture at densities exceeding 10×108 cells/ml of the bed volume (CelliGen, New Brunswick Scientific, Edison, N.J.). However, like other such methods, these devices have high cost and limited applications.
Perfusion operations have tremendous potential for growing the large number of cells needed for human cell and genetic therapy applications.
The central problem in perfusion culture is how to retain the cells in the bioreactor. Prior art can be classified into four basic separation technologies: filtration, gravity sedimentation, centrifugation and continuous perfusion. Filtration methods require some means to keep the filter from clogging over the required weeks of operation. Cross-flow filters are typically used. Here a high tangential liquid velocity is used to keep the surface clean. Spinning filters are another embodiment of this concept. Gravity sedimentation can be used to separate the cells and several types of inclined settlers have been reported. The major problem with settlers is the varying sedimentation characteristics of different cells and the difficulty in scale-up to industrial systems. Centrifugation has found limited application in cell culture due to the difficulty in maintaining sterility.
The first three methods share a common weakness—in that the liquid from the bioreactor must be pumped through the separation device and the cell-enriched material returned to the bioreactor. Keeping this recirculation loop sterile is difficult, and contamination often occurs. To maintain the high cross-flow velocity necessary to prevent clogging, the cells are subjected to high pumping shear in the recirculation loop and are often damaged. Oxygen depletion can also occur if the pumping rate is too slow. These factors often lead to degradation in product quality and quantity. The fourth type of method avoids some of these problems by eliminating the need to use a pump-around loop wherein nutrient media is removed from the bioreactor and replaced with fresh media by filtering the contents of the bioreactor continuously.
However, the method of removing nutrient media from a bioreactor and replacing it with fresh media becomes a difficult process when using thousand of liters of nutrient media and very high density of cell culture, as it is becoming a normal exercise in commercial production. Whether it is placing the filter in a special place in the bioreactor to reduce exposure to high cell titer or gently shaking the filter to keep the filter from getting clogged, these prior art methods are inadequate for large-scale commercial production. Even if the perfusion is performed at a rate of one to two media volume exchanged per day, bioreactors containing thousands of liters with suspension culture at high titer would make it impossible to use any of the current art to accomplish the filtration and harvesting of the nutrient media. Large-scale filtration would require faster passage of media through the filter and that would inevitably bring cell culture in direct contact with filter surface resulting in fouling and blockage of the filters. Unfortunately, the flexible and disposable bioreactors wherein most of the art of perfusion filtration is developed are not suitable for large-scale operations and thus the dearth of technology in the field of cell culture processing was not fully appreciated.
There is therefore a dire need to invent systems useful for any size of operation, ones that could not be blocked regardless of the rate of filtration and ones that could be sterilized and be also affordable. The instant invention resolves all of these problems by utilizing a method scrubbing the filter surface by fine bubbles of, which in itself may help growth of cell culture.